The GeneArt® Synechococcus Protein Expression Kit is a second-generation Synechococcus cloning and expression system from Life Technologies. Like the first-generation GeneArt® Synechococcus Engineering Kit, this expression system employs Cyanobacterium Synechococcus elongatus strain PCC 7942, but is optimized for high-level expression and offers dual protein tags for detection and/or purification of your gene of interest. It includes frozen cyanobacterial cells, expression vector, OneShot® TOP10 Competent E. coli cells, and easy-to-follow protocols. Our Gibco® BG-11 Media, sold separately, is optimized for the growth and maintenance of select Cyanobacteria including S. elongatus.
- Express >10% total soluble protein of your gene of interest
- Detect and purify your gene of interest with 6His TEV and/or V5-His epitope tags
- Use (optional) seamless assembly to create your constructs
- Enables reliable results with exceptional strain viability and purity
Better expression and a choice of purification tags
The MCS vector in the GeneArt® Synechococcus Protein Expression Kit is designed to provide constitutive and robust expression in Synechococcus elongatus. Expression is controlled by promoter psbA1, a ribosome binding site (RBS), and optional initiation and stop codons depending on where you clone your gene. Features of the vector include:
- Strong constitutive promoter, psbA1, for robust expression of recombinant gene of interest
- Ribosome binding site (RBS) for improved expression of some genes
- Targeted integration of your gene of interest into the S. elongatus genome
- N-terminal 6His TEV and C-terminal V5-His epitope tags
- Spectinomycin resistance gene for selection in Synechococcus
- Flexible multiple-cloning site vector
The gene for β-glucuronidase (GUS) was cloned into vector pSyn_6 and transformed into Synechococcus elongatus (negative for GUS activity) following manufacturer's recommended protocols. GUS activity levels produced usind the pSyn_6 vector were more than 100-fold greater than those observed from our first-generation GeneArt® Algae Engineering system (pSyn_1).
The gene for β-glucuronidase (GUS) was cloned into vector pSyn_6 and transformed into Synechococcus elongatus (negative for GUS activity) following manufacturer's recommended protocols. GUS expression was measured by western blot analysis and showed GUS protein represented >20% total soluble protein. TSP = total soluble protein.
Greater than 80% integration efficiency
The transformation of Synechococcus elongatus PCC 7942 relies on homologous recombination between the cell’s chromosome and exogenous DNA that is not autonomously replicating and contains sequences homologous to the chromosome. The location of integration into the chromosome (neutral site, NS1) was developed as a cloning locus (Clerico et al., 2007) as it can be disrupted without any aberrant phenotype, thus allowing recombination of ectopic sequences. When transformed with vectors containing an antibiotic resistance cassette and neutral site sequences, a double homologous recombination event occurs between the neutral site vector and the S. elongatus chromosome. The selective marker (spectinomycin) and the gene of interest driven by a promoter are inserted into the neutral site and the vector backbone (pUC) is lost, allowing the expression of recombinant genes in S. elongatus PCC 7942.
Gibco® BG-11 Media—optimized for Cyanobacteria
Gibco® BG-11 Media, offered separately or in some configurations of our GeneArt® kits, is optimized for the growth and maintenance of select Cyanobacteria including Synechococcus elongatus. The 1X formulation lets you avoid laborious media preparation steps. The award-winning bottle design makes manipulation in the biosafety cabinet easier, minimizes the risk of contamination, and helps you perform cell culture more consistently. Superior packaging and quality, greater reliability, and improved consistency in Cyanobacteria culture results in better overall efficiency and more robust data.
Versatile Cyanobacteria for study or bioproduction
The Cyanobacterium Synechococcus elongatus strain PCC 7942, is an excellent system for studying photosynthesis, circadian rhythms, nutrient regulation, environmental response, and lipid metabolism. Cyanobacteria, also known as blue-green algae, are a common platform for bioproduction of alcohols and chemicals due to their small genome size and the ease with which they can be genetically manipulated by natural transformation.
First-generation Synechococcus Engineering Kits